Abstract

Breast cancer is the most prevalent cancer in women worldwide and mutations in breast cancer susceptible genes, BRCA1 and BRCA2 are the genetic factors conferring highest risk (40-70%) of developing breast cancer. Although BRCA proteins are known to maintain genomic stability mainly by homologous recombination-mediated DNA repair, detailed mechanisms of how BRCA loss induces tumorigenesis remain unclear. Interestingly, while inactivation of both alleles of BRCA1 or BRCA2 genes is necessary for tumor development, their loss in embryonic cells affects cell viability. To explain this paradox, we hypothesize that cells lacking BRCA1 or 2 are able to survive and are predisposed to tumorigenesis due to mutations in other genes such as those involved in cell cycle regulation or DNA damage response. In the current study, we propose to identify such genes using a mouse embryonic stem (mES) cell-based insertional mutagenesis screen. In mES cells with one mutant allele of Brca2 and the other flanked by loxP sites, cell death can be induced by Cre-mediated loss of the conditional allele. To identify genes whose up-regulation can rescue BRCA2 loss-induced cell death, we transduced these mES cells with murine stem cell retrovirus (MSCV) expressing Cre recombinase. We obtained Brca2-null ES cells that were rescued by the viral insertion and used them to clone the viral insertional sites. We identified several candidate genes that were upregulated in viable ES cells. One of these genes encodes a PDZ domain-containing protein, GIPC3. Expression of Gipc3 was elevated 3 to 4-fold in the rescued clone. Furthermore, we rescued the lethality of Brca2-null ES cells by overexpressing Gipc3. The rescued cells were hypersensitive to DNA damaging agents and exhibited an overall increase in genomic instability. Mechanistically, we found DNA damage-induced p21 expression to be attenuated in GIPC3 over-expressing clones. Because p21 is a key mediator of BRCA2 loss-induced cell death, we hypothesize that GIPC3 overexpression may inhibit p21 expression resulting in viable Brca2-null ES cells. We are now studying the detailed signaling pathway of how GIPC3 affects p21 expression. In addition, we are examining if Gipc3 overxpression can rescue the lethality of Brca2ko/ko embryos and contribute to tumorigenesis in Brca2ko/+ mice. We are also examining the expression of GIPC3 in human BRCA2-deficient tumors.